MODERN AGRICULTURAL BIOTECHNOLOGY - ucbiotech...

American Council on Science and Health 1995 Broadway, Suite 202 New York, New York 10023-5882 Tel. (212) 362-7044 • Fax (212) 362-4919 URL: http://www.acsh.org • Email: [email protected]

New and innovative techniques will be required to improve the production and efficiency of the global agriculture sector to ensure an ample supply of healthy food. This challenge is confounded by the inequity between the affluent and developing countries that is likely to continue to widen. It appears that only a handful of technologies are affordable by the least developed countries and are sufficiently scale neutral developed countries and are sufficiently scale neutral to be accessible to poorer countries. In this book the American Council on Science and Health explores the benefits of, and barriers to, biotechnology — one such technology that offers efficient and cost-effective means to produce a diverse array of novel, value-added traits and products.

The American Council on Science and Health is a consumer education consortium concerned with issues related to food, nutrition, chemicals, pharmaceuticals, lifestyle, the environment and health. It was founded in 1978 by a group of scientists concerned that many important public policies related to health and the environment did not have a sound scientific basis. These scientists did not have a sound scientific basis. These scientists created the organization to add reason and balance to debates about public health issues and bring common sense views to the public.

A GUIDE TO MODERN

AGRICULTURAL BIOTECHNOLOGY

FOOD AND YOU

a publication of the

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Food and YouA Guide to Modern Agricultural Biotechnology

Written byMartina Newell-McGloughlin, D.Sc.

Director, UC Systemwide Biotechnology Research and Education Program University of California, Davis

Bruce Chassy, Ph.D. Professor Emeritus, Department Food Science and Human Nutrition

University of Illinois at Urbana-Champaign

Gregory Conko, J.D. Executive Director and Senior Fellow

Competitive Enterprise Institute

A publication of the

American Council on Science and Health 1995 Broadway, Suite 202 New York, New York 10023-5860 Tel. (212) 362-7044 • Fax (212) 362-4919 URL: http://www.acsh.org • Email: [email protected]

Publisher Name: American Council on Science and Health Title: Food and You: A Guide to Modern Agricultural Biotechnology Price: $14.95 Authors: Drs. Martina Newell-McGloughlin and Bruce Chassy, and Mr. Gregory Conko Subject (general): Science and Health Publication Year: 2013 Binding Type (i.e. perfect (soft) or hardcover): Perfect

ISBN: 978-0-9727094-8-4

Food and You: A Guide to Modern Agricultural Biotechnology. Copyright © 2013 by American Council on Science and Health. All rights reserved. No part of this book may be used or reproduced in any matter whatsoever without written permission except in the case of brief quotations embodied in critical articles and reviews. For more information, contact:

Kent Bradford, Ph.D. Distinguished Professor of Plant Sciences

Director, Seed Biotechnology Center University of California, Davis

James I. Burke, Ph.D. Professor and Chair of Crop Science

University College Dublin

Christopher J. Leaver, Ph.D. Fellow of St John’s College

University of Oxford

Alan G. McHughen, D.Phil. CE Plant Biotechnologist

University of California, Riverside

AcknowledgementsThe American Council on Science and Health (ACSH) appreciates the contributions of the

reviewers named below:

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Table of Contents

Abstract ........................................................................................7

1 Introduction .................................................................................7

2 The technologies ......................................................................... 12

3 Food safety and risk assessment ................................................25

4 Applications overview ................................................................36

5 Agronomic traits and sustainability.......................................... 40

6 Renewable resources ..................................................................50

7 Post-harvest characteristics .......................................................52

8 Nutrition .....................................................................................53

9 Barriers to introduction .............................................................65

10 Coexistence ................................................................................. 71

11 Conclusions ................................................................................75

Glossary ......................................................................................77

Sources .......................................................................................95

Table of Contents

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1Introduction

New and innovative techniques will be required to improve the produc-tion and efficiency of the global agriculture sector to ensure an ample supply of healthy food. This challenge is confounded by the inequity between the affluent and developing countries that is likely to continue to widen. It appears that only a handful of technologies are affordable by the least devel-oped countries and are sufficiently scale neutral to be accessible to poorer countries. Biotechnology is one such technology that offers efficient and cost-effective means to produce a diverse array of novel, value-added traits and products.

The first biotechnology products commercialized in agriculture were crops with improved agronomic traits, primarily pest disease resistance and herbi-cide tolerance whose value was of benefit to the farmers but often opaque to consumers. Currently under development are crops with a more diverse set of new traits that can be grouped into four broad areas, each presenting what, on the surface, may appear as unique challenges and opportunities.

The present and near-future focus is on 1) continuing improvement of agronomic traits such as yield and abiotic stress resistance in addition to the biotic stress tolerance of the present generation of crops; 2) optimizing crop plants for use as biomass feedstocks for biofuels and “bio-synthetics”; 3) the introduction of value-added output traits such as improved nutrition and food functionality; and 4) the application of plants as production factories for therapeutics and industrial products. From a consumer perspective the focus on value added traits, especially improved nutrition, is undoubtedly one of the areas of greatest interest.

Abstract

IntroductionThe introduction of in vitro methods for the transfer of genes (or other

DNA sequences) from one organism to another is a powerful genetic tool that has been applied in medicine, agriculture, and the food and chemical indus-tries. The application of genetic engineering techniques, sometimes called the new biotechnology, has led to innovations as varied as the production of new life-saving pharmaceuticals and crops that do not require the use of synthetic insecticides to control insect pests. This paper focuses on the present and future application of genetic engineering techniques for the improvement of agriculture. The introduction of these new molecular techniques represents a paradigm shift in agriculture. As is often the case when new technologies are introduced, concerns have been raised about the safety of novel organisms

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Food and You: A Guide to Modern Agricultural Biotechnology

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produced using biotechnology and new risks that could be associated with their introduction into the environment.

The following paragraphs briefly review the development of the food and agricultural system, and the need for major increases in global agricultural productivity. They also discuss the nature of the changes that occur in plant breeding and describe the potential risks of the new technology from a scientific perspective.

Throughout history, paradigm shifts can often be traced to a convergence of events where chance favors the prepared mind or, in the case of the history of technology, prepared collective minds. Research from the divergent disciplines of molecular evolution and archeology support the conclusion that one of the most significant convergences in the history of modern civiliza-tion occurred in the marshlands created by the Tigris and Euphrates rivers designated by historians as Mesopotamia and largely occupying the modern region of southern Iraq – the “fertile crescent.” The world generally credits the Sumerians, who lived in this region, with the development of civiliza-tion. Although nearly contemporary river valley civilizations also developed in the Nile Valley of Egypt and the Indus Valley of Pakistan, the Sumerians seem to have been the first people to live in cities and to create a system of writing (Whitehouse, 1977). Scientists also regard the “fertile crescent,” an arc linking Iran, Iraq, Syria, Lebanon, Jordan and Israel/Palestine, as the site of the earlier “Neolithic revolution,” when hunter-gatherers first learned to plant crops, then created permanent settlements to cultivate, guard and harvest them. One line of evidence that supports this conclusion is that wild ancestors of the food crops associated with traditional Middle Eastern and European agriculture are native to this region. The general consensus among historians and anthropologists is that by providing a reliable source of food energy that could be stored for long periods of time, carbohydrates were the principle trigger for this birth of civilization. Cereal grasses of this region have long been considered among the first cultivated crops. Adoption of grain crop cultivation has been considered to be a prerequisite for both eastern and western civilization as settled communities required structures in place to manage land and other resources.

In the intervening 10,000 years, many technologies have been developed and used to enhance productivity of that original coterie of cultivated crops and to bring more into the domestic fold. In the latter half of the 20th Century, major improvements in agricultural productivity were largely based on selective breeding programs for plants and animals, intensive use of chemical fertilizers, pesticides and herbicides, advanced equipment develop-ments and widespread irrigation programs. This has been a very successful

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1Introduction

model for raising productivity but is not without cost and consequent unsus-tainable damage to the environment.

In addition, from a global perspective, these advances have been the prerog-ative of more affluent regions. Farmers in developing countries have not had access to many of these technologies and the more capital-intensive methods of improved production (FAO, 2012). During the coming decades, food and agricultural production systems must be significantly enhanced to respond to a number of wide-ranging and far reaching transformations, including a changing and often unpredictable climate, growing world population, increas-ing international competition, globalization, shifts to increased meat consump-tion in developing countries and rising consumer demands for improved food quality, safety, health enhancement and convenience (UK, 2012). In order to feed a predicted population of roughly 9 billion by 2050, the world will have to double its annual agricultural production over the next 25 years, despite having already quadrupled it in the last 50 years. The inequities between the affluent and developing countries must be addressed using technologies that are scalable across these economic imbalances. Of even greater concern is the very immediate state of current global food reserves. In 2012 the United Nations issued an unprecedented warning about the state of global food supplies (Eliasson, 2012). They noted that failing harvests in the United States, Ukraine and other countries in 2012 eroded global food reserves to their lowest level since 1974, when the world’s population was much lower. World grain reserves are so dangerously low that another year of severe weather in the United States or other food-exporting countries could trigger a major hunger crisis. Clearly, unprecedented needs require innovative solutions to ensure an ample supply of healthy food against competing interests, and this can only be achieved by improving the effectiveness of all components of the global agriculture sector. Innovation is essential for sustaining and enhancing agricultural productivity, and this involves new, science-based products and processes that contribute reliable methods for improving quality, productivity and environmental sustainability.

Agriculturalists have applied approaches such as cross breeding, mutation selection and culling those with undesirable characteristics to modify animals and crop plants over the millennia (Chrispeels and Sadava, 2003). All of these methods depend directly on the selection of desirable novel traits that arise from a variety of kinds of DNA mutations; said another way, novel traits are the results of genetic changes. Thus, from a scientific perspective the term “genetically modified organism” is not an accurate descriptor solely of the products of modern biotechnology, as virtually all domesticated crops and animals have been subjected to varying degrees of genetic modification and selection. Over time, and especially during the last century, plant and animal


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breeders expanded the tools of genetic manipulation beyond conventional cross breeding to use a variety of other breeding techniques. In the case of plants, these tools include aneuploidy, polyploidy, embryo rescue, protoplast fusion, somaclonal variation, anther culture, colchicine for chromosome doubling and mutation breeding through the use of either radiation or chemi-cals (Chrispeels and Sadava, 2003).

Crops developed using the methods described above are common through-out the food chain. For example, seedless banana and watermelon varieties were developed using aneuploidy to triple the number of chromosomes. Bread wheat, developed thousands of years ago, is an allopolyploid plant, containing six entire sets of chromosomes from three different species. Broccoflower was developed using embryo rescue, and male sterility in cauliflower was produced by fusing together protoplasts of radish and cauliflower. Many common tomato varieties are the result of wide crosses between domesticated tomato and wild relatives known to contain high levels of glycoalkaloid toxins. Common varieties of Asian pear, grapefruit and durum pasta wheat were developed with irradiation, or “mutation” breeding for fungal resistance in the former and modified starch in the latter (Newell-McGloughlin, 2008).

Innovations such as these have been essential for sustaining and enhancing agricultural productivity in the past and will be even more important in the future. Innovation is necessary to develop new, science-based products and processes that contribute reliable methods for improving quality, productiv-ity and environmental sustainability. Biotechnology has introduced a new dimension to such innovation, offering efficient and cost-effective means to produce a diverse array of novel, value-added products and tools. It has the potential to improve qualitative and quantitative aspects of food, feed and fiber production, reduce the dependency of agriculture on chemicals (a transi-tion from “chemical solutions to biological solutions”) and slow the increase in the cost of raw materials, all in an environmentally sustainable manner.

Many of the products we eat or wear can be made using the tools of bio-technology. It is possible to enhance the nutritional content, texture, color, flavor, growing season (time to flowering), yield, disease or pest resistance and other properties of production crops. Transgenic techniques can be applied to farmed animals to improve their growth, fitness and other quali-ties. Enzymes produced using recombinant DNA methods (in microorgan-isms/bacteria and yeasts/fungi, etc.) are used to make cheese, keep bread fresh, produce fruit juices and wines and treat fabric for blue jeans and other denim clothing. Other recombinant DNA enzymes are used in laundry and automatic dishwashing detergents.

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1Introduction

We can also engineer recombinant microorganisms to improve the quality of our environment. In addition to the opportunities for a variety of new products, including biodegradable products, bioprocessing using engineered microbes offers new ways to treat and use wastes and to use renewable re-sources for materials and fuel. Instead of depending on non-renewable fossil fuels, we can engineer organisms to convert maize and cereal straw, forest products and municipal waste and other biomass to produce fuel, plastics and other useful commodities. Naturally-occurring microorganisms are being used to treat organic and inorganic contaminants in soil, groundwater and air by a process known as bioremediation. This application of biotechnology has created an environmental biotechnology industry important in water treat-ment, municipal waste management, hazardous waste treatment, bioremedia-tion and other areas.

Used effectively, biotechnology has enormous potential to improve the quality of our life and our environment. This paper focuses a set of technolo-gies that can be grouped together under the rubric modern biotechnology or new biotechnology. It explains briefly what these tools are and how these tools are used to improve crops. The present day applications of these new technologies are described with particular emphasis on transgenic crops – often referred to as genetically modified organisms (GMOs) or genetically-engineered (GE) organisms. Current research and future developments are highlighted. Regulation, public acceptance and barriers to adoption round out the discussion.

This paper provides an overview of the tools, techniques and processes that comprise modern molecular biotechnology, with a primary focus on the genetic modification of plants. It is not meant to be a comprehensive review of all the available technologies or the pros and cons of their current applications, but it is intended to offer a guide, or reference work, beyond the mere basics. Section 2 introduces many of the technologies that have been developed for precisely modifying plants at the molecular level, including both those routinely used by plant genetic engineers and a few whose use has only recently been introduced in this field and others whose application will continue to evolve. Section 3 provides a deeper look at the safety of modified plants for the environment and for use in food and livestock feed. Importantly, it puts the safety of genetically engineered plants into the context of what we’ve learned over thousands of years of plant genetic modification, and it explains why plant biologists are so confident in the potential benefits of the newer technologies.

Section 4 reports on the types of traits that have been introduced into crop plants using recombinant DNA techniques, and it offers a preview of traits that have been or are now being developed. Section 5 summarizes how the


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current generation of biotech crop plants is being deployed and the agro-nomic, economic and environmental benefits that growers have reaped from these products, while also anticipating benefits that may be enjoyed from the next generation of biotech varieties. Sections 6, 7 and 8 look at downstream benefits – those already seen as well as those projected – for consumers, such as new sources of energy and fuels, reduced loss of foodstuffs after harvest to spoilage and pests and increased nutritional value.

Section 9 examines some of the hurdles biotech crop developers, seed breeders and farmers have faced when introducing new varieties, while section 10 looks at ways of ensuring peaceful coexistence between growers who choose biotech crop varieties and those who wish to remain “GE-free.” On the latter issue, there is considerable discussion as to the most appropriate terminology to use for the general class of the products of modern molecular biological techniques, primarily recombinant DNA technology, with the terms genetically modified organisms, GMOs, biotech crops, etc., used somewhat interchangeably. We will primarily use GE.

This paper does not discuss in detail agricultural and environmental risk assessment and regulation. The impact of GE crops on agriculture, agricul-tural sustainability and the environment are briefly described in the text.

The TechnologiesIn the simplest and broadest sense, biotechnology is a series of enabling

technologies that involve the manipulation of living organisms or their sub-cellular components to develop useful products and processes. The capacity to manipulate the genetic makeup of living organisms with complexity and precision has become one of the cornerstones of modern biotechnology. It enables developers to enhance the ability of an organism to produce a par-ticular chemical product (e.g., penicillin from a fungus), to prevent it from producing a product (e.g., ethylene in plant cells) or to enable it to produce an entirely new product (e.g., chymosin in microorganisms).

Most of the fundamental technologies that fall within the broad rubric of biotechnology are well known. The most prevalent include recombinant DNA technology; high throughput sequencing; DNA microarrays; RNA interfer-ence; genomics, proteomics, metabolomics and bioinformatics; and derivative technologies that use the tools of biotechnology, such as marker-assisted selection and novel haploid generation. Before focusing on these technologies, a brief description of a few of the more fundamental tools of biotechnology research is appropriate.

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Recombinant DNA technologyThe steps involved in recombinant DNA technology are: (1) to identify the

gene that directs the production of the desired substance, (2) to isolate the gene using restriction endonucleases/enzymes, (3) to insert the gene with ap-propriate regulatory DNA sequences that control expression of the gene into a suitable DNA molecule (vector) for transformation and (4) to transfer the recombined DNA into the appropriate host organism, generally by transform-ing dozens of single cells in culture. The final step is (5) to select, using a selectable marker gene incorporated in the vector, the transformed cell or cells that have the most desirable characteristics and propagate one or more whole plants from those cells.

Plant transformation is typically accomplished by using either Agrobacterium tumefaciens or a gene gun (see Figure). Agrobacterium tumefaciens is a bacterium that occurs in nature and which causes crown gall disease in many species of plants. It contains a small circular piece of non-chromosomal DNA called a Ti plasmid (Ti for tumor inducing). When this bacterium infects susceptible plants, the Ti plasmid enters cells of the host plant, and specific regions of the Ti plasmid insert themselves into the host cell’s genome. This insertion occurs in a region of the DNA strand with a specific sequence. The host cell then expresses the gene from the bacteria,


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which induces massive cell growth and the plant tumors after which the bacterium is named.

Biotechnology utilizes this natural transformation process by removing the bacterial genes from the region transferred to the host genome and substitut-ing the gene(s) of interest (see part A of Figure). Agrobacterium use for trans-formation was initially limited to use in certain dicotyledonous, or broad-leaf, plants because the wild-type microbe tends only to infect those species. The system has since been improved in the laboratory to allow agrobacterium-me-diated transformation of most major crops, including monocotyledonous, or “grassy,” plants. But agrobacterium is still mainly used to transform dicots.

The other transformation process involves coating tiny gold, tungsten or other heavy metal particles with genes of interest. The coated particles are shot into single cells of the plant of interest using compressed air or another propellant. This is commonly referred to as particle bombardment/accelera-tion, biolistics or the gene gun approach. In a process not fully understood, the transgene(s) are incorporated into a DNA strand of the host genome (see part B of Figure). This process is inefficient but does not have the host species limitation of agrobacterium.

After transformation with either the agrobacterium or gene gun processes, untransformed cells must be eliminated. This is facilitated with the use of selectable marker genes. In the case of an herbicide tolerance gene, the her-bicide tolerance trait itself serves as the selectable marker, since the herbicide will kill non-transformed cells (see part C of Figure). When another trait of interest is being transformed in the crop, a selectable marker, like antibiotic resistance or herbicide tolerance, is used in addition to the primary gene of interest. The cells in culture are treated with the herbicide or an antibiotic, and only those cells that were transformed with the selectable marker genes will survive. Whole plants are then regenerated from the surviving cells by growth on an appropriate medium containing plant hormones.

Following transformation and plant regeneration, the transgenic plants are first extensively tested in the laboratory under containment conditions before extensive testing in field in a range of geographical locations to ensure that the transgene functions properly and confers the desired trait. Not all transgenic plants will express the trait or gene product properly or stably, and these are eliminated before field testing. Once a transgenic plant with robust and stable trait expression has been identified, the trait can then be bred using conventional plant breeding methods into elite crop cultivars best suited to the environmental conditions where the crop is grown.

Microarrays (biochips)DNA microarrays, also commonly known as biochips or DNA chips,

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were developed in the late 1980s and early 1990s primarily by the company Affymetrix. Biochips are a “massively parallel” genomic technology. They facilitate high throughput analysis of thousands of genes simultaneously and are thus a potentially very powerful tools for gaining insight into the complexi-ties of higher organisms including analysis of gene expression, detecting genetic variation, making new gene discoveries, “fingerprinting strains” and developing new diagnostic tools. These technologies permit scientists to conduct large-scale surveys of gene expression in organisms, thus adding to our knowledge of how they grow and develop over time or respond to various environmental conditions. These techniques are especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner. This technology is now largely superseded by next generation sequencing.

RNA silencingIncreased production of desirable characteristics present in crops is a

common goal for breeders – for example, grains with increased protein content and nutritional quality, fruits and vegetables with enhanced nutritional value and flowers with deeper colors. It was in pursuit of the latter goal that a most bemusing and ultimately valuable phenomenon was first observed. While attempting to create “black” petunias as a model for one of the ultimate floricul-ture aspirations, the “black” rose, Jorgensen et al. (1996) of UC Davis attempted to over-express the chalcone (a pigment precursor) synthesis gene by introduc-ing a modified copy under a strong promoter. The surprising result was white flowers, and many strange variegated variations between purple and white. This was the first demonstration of what has come to be known as post-tran-scriptional gene silencing (PTGS). While initially it was considered a strange phenomenon limited to some plant species, it is now recognized to be a signifi-cant regulatory mechanism in all higher organisms. RNA interference (RNAi) in animals and basal eukaryotes, “quelling” in fungi and posttranscriptional gene silencing (PTGS) in plants are examples of a broad family of phenomena collectively called RNA silencing (Hannon 2002; Plasterk 2002). In addition to its occurrence in these organisms, it has roles in viral defense and transposon silencing mechanisms, among other things. Perhaps most exciting, however, is the emerging use of PTGS and, in particular, RNA interference (RNAi) – PTGS initiated by the introduction of double-stranded RNA (dsRNA) – as a tool to “knock out” expression of specific genes in a variety of organisms.

Instead of producing large quantities of new proteins, high-expressing transgenes (genes from another source) introduced into the plant can actually inhibit the expression of the plant’s own genes by triggering sequence-specific destruction of similar transcripts (Dykxhoorn et al., 2003). Thus, the trans-gene ends up silencing both its own expression and that of similar endog-enous genes when the concentration of transgene transcript (mRNA) becomes


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too high in the cytoplasm. This unintended “RNA silencing” can nonetheless be harnessed by scientists, for example to eliminate unwanted gene expres-sion, and is used by the plant itself to inhibit protein synthesis by infecting RNA viruses. A significant example of this (which was determined after the fact) is coat-protein-gene protection introduced into papaya to protect against papaya ring spot virus, one of the few transgenic gene-silencing systems in commercial production (Fitch et al., 1992). Recent studies have demon-strated that RNAi-mediated mechanisms and PTGS have been unknowingly exploited for many years by plant breeders in many new crop phenotypes (Parrott et al., 2010). Parrott et al. concluded that crops modified using small RNA technology that produce no novel proteins or metabolites pose little if any novel hazards to food safety or the environment and that a subset of the currently applied regulatory paradigm can be used for their safety assessment.

RiboswitchesEach cell must regulate the expression of hundreds of different genes in

response to changing environmental or cellular conditions. The majority of these sophisticated genetic control factors are proteins, which monitor metab-olites and other chemical cues by selectively binding to targets. RNA also can form precision genetic switches and these elements can control fundamental biochemical processes.

Riboswitches are a type of natural genetic control element that uses an untranslated sequence in an mRNA to form a binding pocket for a metabolite that regulates expression of that gene. Potentially engineered riboswitches might function as designer genetic control elements.

Protein engineeringAnother area of genetic engineering, in the broader sense, is protein

engineering (Brannigan and Wilkinson, 2002). New enzyme structures may be designed and produced in order to improve on existing enzymes or create new activities. The principal approaches are: 1) site-directed mutagenesis (oligonucleotide-directed mutagenesis), 2) random mutation and selection and 3) directed evolution, which is a refinement of the latter. However, from a practical point of view, much of the research effort in protein engineering has gone into studies concerning the structure and activity of enzymes chosen for their theoretical importance or ease of preparation rather than industrial relevance. With a greater focus on “green” production systems, this emphasis is now shifting.

Genomics, proteomics, metabolomics and bioinformaticsFunctional genomics can be defined as establishing a link between gene

expression and cellular function (Prevsner, 2009). Thousands of genes in

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a single tissue type vary in levels of expression at different developmental stages, in health and disease, at different chronological times and in response to environmental variation. Functional genomics provides insight into all the genes involved and the roles they play. Although applications in medicine have been the main thrust of this development, this technology is now having a major impact in agricultural biotechnology, and most specifically plant biotechnology research.

The proteome is the complete set of proteins expressed and modified after their expression from the genome. Proteomics refers to the study of the structure, function, location and interaction of proteins within and between cells. Proteomics is particularly being driven by global analyses of gene expression and inferences derived from DNA sequence data. However, the study of proteins is not a simple linear extrapolation from knowledge of the DNA sequence. It is a highly complex multidimensional field of endeavor. Each cell produces thousands of proteins, each with a specific function. Proteins differ greatly from one another, even within the same individual, but DNA molecules are remarkably similar. In addition, unlike the unvarying genome, an organism’s proteome is so dynamic that an almost infinite variety of protein combinations exist. The proteome varies from tissue to tissue, cell to cell, and with age and time. The cellular proteome changes in response to other cells in the body and to external environmental stimuli. Methods for analysis of protein profiles and cataloging protein-protein interactions on a genome-wide scale are technically more difficult but are improving rapidly, especially for microbes. Functional genomics will impact most areas of biology, from fundamental biochemistry to improvement of quality, and agronomic traits in crops, improved protection against pathogenic microbes, and improved exploitation of beneficial microbes.

Sequencing of organismal genomes has created a vast quantity of data, which is not easily examined or understood. In many ways plant sequences are more complex than even the human genomes given the sheer amount of DNA. Similar problems exist for a wide variety of topics in functional genomics, proteomics, metabolomics and so on, primarily due to the scale and parallel nature of these approaches.

The construction of relational databases, as well as the development of efficient methods for searching and viewing these data, constitutes a disci-pline called “bioinformatics.” In a broader view, bioinformatics contains computational or algorithmic approaches to the production of information from large amounts of biological data, and this might include prediction of protein structure, dynamic modeling of complex physiological systems or the statistical treatment of quantitative traits in populations to determine the genetic basis for these traits. Unquestionably, bioinformatics is now an


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essential component of all research activities utilizing structural and func-tional genomics approaches for analysis at the sequence level, in structure modeling, and in modeling, linking and simulating complex higher-level structures such as metabolic and neurological pathways.

Evolving techniquesAs with any rapidly evolving field tools and techniques are being improved

at a rapid pace. A brief overview will be provided here. This not intended to be either comprehensive or complete review but serves as an example of the rapid development of new molecular tools.

Genome editingFigure 2. Genome editing tools (source: Addgene https://www.addgene.org/CRISPR/guide/)

One of the subtler modification systems, which will provide a challenge for regulatory oversight, is what may be referred to as next generation directed mutagenesis. Genome editing, specifically genome editing with engineered nucleases (GEEN), is a form of fined-tuned gene modification using novel nucleases which results in minor insertions, replacements or deletions in a highly targeted manner. There are a number of gross categories of

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engineered nucleases, mostly based, like the original restriction endonucle-ases, on immune systems from their source organisms: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and homing endonucleases including engineered hybrid meganucleases. These nucleases create targeted specific double-stranded breaks at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end-joining (NHEJ) (Esvelt and Wang, HH., 2013).

Genome editing with these nucleases is different from silencing the gene of interest by short interfering RNA (siRNA) in that the engineered nuclease is able to modify DNA-binding specificity and therefore can, in principle, cut any targeted position in the genome and introduce modification of the endogenous sequences for genes that are impossible to specifically target by conventional RNAi. These and evolving editing tools have tremendous potential to intro-duce very targeted modifications, which will call into question the notion of “genetic engineering” and will present a problem for regulatory authorities, if they, without justification, decide they need to capture them for review. It will be, for all intents and purposes, impossible to detect, so enforcing oversight will be prove to be a challenge.

Other systems, such as synthetic biology (the design and construction of new biological parts, devices and systems) and genome-scale engineering are being enabled through advances in high-throughput DNA sequencing, and large-scale biomolecular modeling of metabolic and signaling networks will contribute to food and agriculture production systems in the future.

Metabolic engineering One field of great interest is the modification of complex traits – traits not

necessarily associated with a single inserted gene – especially those that may have an epigenetic component.

Plants produce between 200 and 250,000 secondary metabolites. Analysis of these metabolites (most specifically metabolomic analysis) is a valuable tool in better understanding what has occurred during crop domestication (lost and silenced traits) and in designing new paradigms for more targeted crop improvement that is better tailored to current needs. In addition, with modern techniques, it confers the potential to seek out, analyze and introgress traits of value that were limited in previous breeding strategies. Research to improve the nutritional quality of plants has historically been limited by a lack of basic knowledge of plant metabolism and the challenge of resolv-ing complex interactions of thousands of metabolic pathways. Metabolic


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engineering is generally defined as the redirection of one or more reactions (enzymatic and otherwise) to improve the production of existing compounds, produce new compounds or mediate the degradation of undesirable com-pounds. It involves the redirection of cellular activities by the modification of the enzymatic, transport and/or regulatory functions of the cell. Significant progress has been made in recent years in the molecular dissection of many plant pathways and in the use of cloned genes to engineer plant metabolism. So, in sum, a complementarity of techniques both traditional and novel is needed to metabolically engineer plants to produce desired traits.

Although progress in dissecting metabolic pathways and our ability to manipulate gene expression in genetically-engineered (GE) plants has pro-gressed apace, attempts to use these tools to engineer plant metabolism have not quite kept pace. Since the success of this approach hinges on the ability to change host metabolism, its continued development will depend critically on a far more sophisticated knowledge of plant metabolism, especially the nuances of interconnected cellular networks, than currently exists. This complex interconnectivity is regularly demonstrated. Relatively minor genomic changes (point mutations, single-gene insertions) are regularly observed following metabolomic analysis to lead to significant changes in biochemical composition. However, what on the surface would appear to be other, more significant genetic changes unexpectedly yield little phenotypical effect.

A number of new approaches are being developed to counter some of the complex problems in metabolic engineering of pathways. Such approaches include use of RNA interference to modulate endogenous gene expression or the manipulation of transcription factors (Tfs) that control networks of metabolism (Bruce et al., 2000; Gonzali, Mazzucato, & Perata, 2009; Kinney AJ, 1998). For example, expression in tomatoes of two selected transcription factors (TFs) involved in anthocyanin production in snapdragon (Antirrhinum majus L.) led to accumulation of high levels of these flavonoids throughout the fruit tissues, which, as a consequence, were purple. Such expres-sion experiments hold promise as an effective tool for the determination of transcriptional regulatory networks for important biochemical pathways (Gonzali S et al., 2009). Gene expression can be modulated by numerous transcriptional and posttranscriptional processes. Correctly choreographing the many variables is the factor that makes metabolic engineering in plants so challenging.

In addition, there are several new technologies that can overcome the limitation of single gene transfers and facilitate the concomitant transfer of multiple components of metabolic pathways. One example is multiple-trans-gene direct DNA transfer, which simultaneously introduces all the compo-nents required for the expression of complex recombinant macromolecules into the plant genome as demonstrated by a number including Nicholson

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et al. (2005), who successfully delivered four transgenes that represent the components of a secretory antibody into rice (Carlson et al., 2007), construct-ing a minichromosome vector that remains autonomous from the plant’s chromosomes and stably replicates when introduced into maize cells. This work makes it possible to design minichromosomes that carry cassettes of genes, enhancing the ability to engineer plant processes such as the produc-tion of complex biochemicals.

It was demonstrated (Christou P, 2005) that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants stably expressing several different transgenes. Since no vector back-bones are present, this technique allows the construction of transformants that contain only the DNA sequences required to produce the desired new trait. They used combinatorial direct DNA transformation to introduce multi-complex metabolic pathways coding for beta carotene, vitamin C and folate. They achieved this by transferring five constructs controlled by different endosperm-specific promoters into white maize. Different enzyme combina-tions show distinct metabolic phenotypes resulting in 169-fold beta carotene increase, six times the amount of vitamin C and a doubling of folate produc-tion, effectively creating a multivitamin maize cultivar (Naqvi et al., 2009). This system has an added advantage from a commercial perspective in that these methods circumvent problems with traditional approaches, which not only limit the amount of sequences transferred but may disrupt native genes or lead to poor expression of the transgene, thus reducing both the numbers of transgenic plants that must be screened and the subsequent breeding and introgression steps required to select a suitable commercial candidate.

“Omics”-based strategies for gene and metabolite discovery, coupled with high-throughput transformation processes and automated analytical and functionality assays, have accelerated the identification of product candi-dates. Identifying rate-limiting steps in synthesis could provide targets for modifying pathways for novel or customized traits. Targeted expression will be used to channel metabolic flow into new pathways, while gene-silencing tools will reduce or eliminate undesirable compounds or traits or switch off genes to increase desirable products (Davies, 2007; Herman, Helm, Jung, & Kinney, 2003; Liu, Singh, & Green, 2002). In addition, molecular marker-based breeding strategies have already been used to accelerate the process of introgressing trait genes into high-yielding germplasm for commercialization. Table 1 summarizes the work done to date on specific applications in the categories listed above.

More specific example of technology that applies for complex modifications is described in the relevant applications below.


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Trait Crop (trait detail) Reference

Protein and amino acidsProtein quality

and levelBahiagrass (protein↑) Luciani et al. 2005

Canola (amino acid composition)

Roesler et al. 1997

Maize (amino acid composi-tion; protein↑)

Cromwell 1967, 1969;Yang et al. 2002; O’Quinn et al. 2000; Young et al. 2004

Potato (amino acid composi-tion; protein↑)

Chakraborty et al. 2000; Li et al. 2001; Yu and Ao 1997; Atanassov et al. 2004

Rice (protein↑; amino acid ) Katsube et al. 1999

Soybean (amino acid balance) Rapp 2002; Dinkins et al. 2001

Sweet Potato (protein↑) Prakash et al. 2000

Wheat (protein↑) Uauy et al. 2006

Essential amino acids

Canola (lysine↑) Falco et al. 1995

Lupin (methionine↑) White et al. 2001

Maize (lysine↑; methionine↑) Agbios 2006; Lai and Messing 2002

Potato (methionine↑) Zeh et al. 2001

Sorghum (lysine↑) Zhao et al. 2003

Soybean (lysine↑; tryptophan↑) Falco et al. 1995; Galili et al. 2002

Oils and Fatty AcidsCanola (lauric acid↑; γ-linolenic acid↑; + ω-3 fatty acids; 8:0 and 10:0 fatty acids↑; lauric + myristic acid↑; oleic acid↑)

Del Vecchio 1996; Froman and Ursin 2002; James et al. 2003; Ursin, 2003, Dehesh et al. 1996; Agbios 2006; Roesler et al. 1997

Cotton (oleic acid↑; oleic acid + stearic acid↑)

Chapman et al. 2001; Liu et al. 2002

Linseed (+ ω-3 and- 6 fatty acids) Abbadi et al. 2004

Table 1 – Examples of Crops in Research with Nutritionally Improved Traits1

1 Excludes protein/starch functionality, shelf life, taste/aesthetics, fiber quality and allergen reduction traits. Modified from Newell-McGloughlin,2008 (25)

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The Technologies 2


Maize (oil↑) Young et al. 2004

Oil Palm (oleic acid↑ or stearic acid↑; oleic acid↑ + palmitic acid↓)

Parveez 2003; Jalani et al. 1997

Rice (α-linolenic acid↑) Anai et al. 2003

Soybean (oleic acid↑; γ-linolenic acid↑Stearidonic Acid↑)

Kinney and Knowlton 1998; Reddy and Thomas 1996, SDA, 2011

Safflower (γ Linoleic Acid GLA↑) Arcadia, 2008

CarbohydratesFructans Chicory, (fructan↑; fructan

modification)Smeekens 1997; Sprenger et al. 1997 Sévenier et al (1998)

Maize (fructan↑) Caimi et al. 1996

Potato (fructan↑) Hellwege et al. 1997

Sugar beet (fructan↑) Smeekens 1997

Frustose, Raffinose, Stachyose

Soybean Hartwig et al 1997

Inulin Potato (inulin↑) Hellwege et al. 2000

Starch Rice (amylase ↑) Chiang et al. 2005, Schwall, 2000

Micronutrients and functional MetabolitesVitamins and Carotenoids

Canola (vitamin E↑) Shintani and DellaPenna 1998

Maize (vitamin E↑ ; vitamin C↑; beta-carotene↑; folate↑)

Rocheford 2002; Cahoon et al. 2003; Chen et al. 2003; Naqvi et al. 2009

Mustard (+β-carotene) Shewmaker et al. 1999

Potato (β-carotene and lutein↑) Ducreux et al. 2005

Rice (+ β-carotene) Ye et al. 2000

Strawberry (vitamin C↑) Agius et al. 2003

Tomato (folate↑; phytoene and β-carotene↑; lycopene↑; provitamin A↑)

Della Penna, 2007, Díaz de la Garza et al. 2004; Enfissi et al. 2005; Mehta et al. 2002; Fraser et al. 2001; Rosati 2000


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Functional 2ndrymetabolites

Apple (+stilbenes) Szanowski et al. 2003

Alfalfa (+resveratrol) Hipskind and Paiva 2000

Kiwi (+resveratrol) Kobayashi et al. 2000

Maize (flavonoids↑) Yu et al. 2000

Potato (anthocyanin and alkaloid glycoside↓; solanin↓)

Lukaszewicz et al. 2004

Rice (flavonoids↑; +resveratrol) Shin et al. 2006; Stark-Lorenzen 1997

Soybean (flavonoids↑) Yu et al. 2003

Tomato (+resveratrol; chlo-rogenic acid↑; flavonoids↑; stilbene↑anthocynanins↑)

Giovinazzo et al. 2005; Niggeweg et al. 2004; Muir et al. 2001; Rosati, 2000, Gonzali et al, 2009

Wheat (caffeic and ferulic acids↑; +resveratrol)

UPI 2002

Mineral availabilities

Alfalfa (phytase↑) Austin-Phillips et al. 1999

Lettuce (iron↑) Goto et al. 2000

Rice (iron↑) Lucca et al. 2002

Maize(phytase↑, ferritin↑) Drakakaki 2005, Han, 2009

Soybean (phytase↑) Denbow et al. 1998

Wheat (phytase↑) Brinch-Pedersen et al. 2000

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Food safety and risk assessment 3

Food safety and risk assessment 3The nature of genetic changes in crops

Historically, agriculturists, and more recently plant breeders, selected improved crops based on changes that arose as a result of genetic modifica-tion (naturally occurring mutations) of DNA without any knowledge of the nature of the molecular modifications that had occurred in the DNA or result-ing changes in the content of proteins and metabolites contained in newly selected varieties. The introduction of high throughput DNA sequencing methods coupled with bioinformatic analysis, as well as improved methods for evaluating/analyzing the proteome and metabolome of crop plants, has provided insight into the molecular changes that occur as a result of plant breeding. The kinds of DNA modifications that are associated with classi-cal plant breeding or through transgene insertion have been assessed and compared (see for example Parrott et al., 2010; Weber et al., 2012).

There now exists a significant body of evidence that demonstrates that all forms of plant breeding introduce a variety of changes in DNA, ranging from point mutations and single base pair deletions and insertions, loss or acquisition of genes, to changes in numbers of whole chromosomes. When compared to classical plant breeding methods, transgene insertion has been observed to produce less unintended DNA modification. Studies have also shown that transgenic crop varieties more closely resemble their parental lines than do other varieties of the same crop with respect to their proteomic and metabolomics profiles (Ricroch et al., 2012). It should not be overlooked, however, that in most plant breeding programs successive rounds of planting and selection are used to cull out events with obvious and/or undesirable phenotypes and select for plants with unchanged/superior agronomic and phenotypic traits. Regardless of the method of breeding applied to select for genetic changes, the candidate plants that are advanced for potential distribu-tion and planting should closely resemble their parental lines – with the sole exception of the intended modification. It also appears that environmental and cultural conditions have more impact on plant composition than do breeding and selection programs (Ricroch et al., 2012).

In light of these substantial and unpredictable genetic modifications, which occur in crop plants that are common in the human diet, the comparatively simple and more precise modifications performed with recombinant DNA techniques – what we know as modern biotechnology – appear to be unique only in the breeder’s improved ability to control the resulting phenotype of the modified cultivars. As long ago as 1987, an analysis published by the National Academy of Sciences (USA) examined the available research and concluded that plants and other organisms produced using genetic engineering techniques


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pose no new or different risks to human health or the environment than those produced using other breeding methods (NAS, 1987). Since that time, the National Academies, the EU and the governments of a number of countries have on several occasions reviewed the scientific literature on the safety of biotech crops, and each time they have reached the same conclusion.

Transgenic crops produced using the new biotechnology are also regulated by governments and may not be released to farmers or consumers until they have successfully passed a rigorous pre-market safety assessment (Kok and Kuiper, 2003; König et al., 2004; Codex, 2003). On a case-by-case basis, the safety assessor seeks to determine if the new trait introduced into a crop is cause for safety concerns. In principle, the focus of regulators is on the safety of the new trait and not on the fact that genetic engineering has been used to introduce the new trait. Yet, paradoxically, crops developed using less precise and more disruptive methods of breeding may be released without any pre-market regulatory review.

The safety assessor’s role is to ensure that the new crop variety is as safe as, or safer than, other varieties of the same crop. Safety review does not, and cannot, prove that a crop is absolutely without risk because, as we discuss below, no food is completely without risk or risk of being used in an unsafe way. As noted above, all breeding produces changes in DNA that could produce unintended hazards (Cellini et al., 2004). There is unfortunately much confusion on this specific issue, with critics of transgenic crops claiming that the use of modern biotechnology is unsafe per se. A major source of misunderstanding about the safety of biotech crops stems from the belief that the crops produced around the world today are totally innocuous and that any genetic modification could adversely affect safety.

Are crop plants produced by “conventional” plant breeding absolutely safe to consume? Many crops produce potentially toxic phytochemicals to protect themselves from pests. Potatoes and tomatoes, for example, naturally contain low levels of potentially lethal glycoalkaloid toxins, solanine and chaconine. Rapeseed, the cultivated plant from which canola was derived, contains both toxic erucic acid and anti-nutrient glucosinolates. And kidney beans contain phytohaemagglutinin in levels sufficient to be toxic if undercooked. Cassava (Manihot esculenta), a major staple crop in many developing countries, contains cyanogenic glycosides in sufficient quantity to cause death if not properly processed to remove cyanide; chronic low-level consumption can cause goiter. A few of the most commonly eaten plants give rise to food allergies (e.g., peanuts and other groundnuts, tree nuts, soybean, wheat, kiwi fruit and sesame). And an estimated 99 percent or more (by weight) of the pesticides that humans consume in food are chemicals that plants produce naturally to defend themselves from predators. Few of these have been fully

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tested for human safety. But roughly half of those that have been tested were found to be rodent carcinogens (Ames, Profet, & Gold, 1990).

The natural toxins and other potentially hazardous compounds present in dozens of common food plants can be accidentally raised to harmful levels with basic hybridization plant breeding. And other techniques, such as embryo rescue and mutation breeding could cause unpredictable genetic changes that raise similar risks. These unanticipated effects are, however, less likely to occur with biotechnology breeding. Indeed, biotechnology approaches can be employed to downregulate or even eliminate the genes involved in the metabolic pathways for the production and/or activation of such plant toxins and also allergens such as globulins in peanuts (Dodo, 2008). Ironically, many of our daily staples would be banned if subjected to the rigorous safety and testing standards applied to crop plants modified using recombinant DNA technology.

The older, non-biotech breeding techniques allow for no control at the genome level. Rather, multiple genes are transferred together or mutated and unwanted traits are eliminated through subsequent selection and backcross-ing. Plants created by these conventional phenotypic selection techniques undergo no formal food or environmental safety evaluation prior to introduc-tion into the environment and marketplace, other than normal agricultural variety testing. This is not to suggest that classical breeding methods are inherently unsafe. Despite the extensive genetic modification of crop plants by these diverse methods, cases of novel or completely unexpected adverse consequences for commercialized varieties of these crops are extremely rare (e.g., high glycolalkaloid Lenape potato).

Nor should we be concerned that biotechnology methods permit breeders to move genes between unrelated taxonomic kingdoms. The recent massive accumulation of DNA sequencing data shows extensive genetic similarity among genomes of diverse organisms that are only remotely related. For example, parts of the nucleic acid sequence of a common bacterium present in our guts, Escherichia coli, have been found in the DNA of organisms such as oilseed rape, amphibians, birds, grasses and mammals – including humans. Such findings put in doubt the value of assigning genes to a particular species and the validity of using terms such as “species-specific” DNA. Due to the common genetic ancestry of all living organisms, there already is broad sharing of identical or very similar genes across taxonomic kingdoms, so there is nothing inherently unnatural or unsafe about moving a gene from bacteria or viruses into a crop plant. Modern biotechnology would not in fact be possible if genes did not function in the same way in all organisms. In particular, the genes that encode proteins that are involved in functions that are common to all cells (for example: DNA replication, RNA transcription


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and protein synthesis) are similar in all organisms. The safety of any genetic modification depends solely on the function of the specific gene or genes that are moved, how they are expressed in the new host organism and the impact that new phenotype has on the modified organism’s environment and use. Genes are not organism specific – harmful genes may be moved with simple hybridization between closely related plant species, and helpful or benign genes may be moved between kingdoms to no ill effect.

Based on the foregoing discussion it should be clear that all new varieties of crops are the result of genetic modification regardless of the technology used for their development. To date, new crop varieties have been almost without exception safe to plant and safe to consume. The small number of document-ed cases in which a new variety was found to be unsafe for consumers were all the products of classical breeding methods (NRC 2004). Nevertheless, new varieties have proven so comparatively safe that non-biotech ones are released to farmers with essentially no oversight by regulators and, with very few exceptions, no requirements for safety testing. Crops produced using modern biotechnology are, however, all subject to special regulation with associated significant costs implications.

There has been great misunderstanding about why pre-market safety assessment should be required of these crops. It is often asserted that genetic modification may produce unforeseen and unintended changes in crops, but as we have seen all breeding produces unintended changes (see above and Cellini et al., 2004; Parrott, 2010), so that cannot be the scientific basis for regulating these crops. Nevertheless, aspects of the regulatory framework in every country that permits the commercial use of biotech crops, or food and animal feeds derived from them, are premised on the belief that unique risks arise from the transformation process itself. Each time a gene is introduced into a plant, the resulting organism (or “transformation event”) is treated as a unique product for the purposes of regulation. Even if copies of a single gene encoding the same protein are inserted into different plants of the same species, each result-ing transformation event must be tested and approved separately.

The rationale for such event-specific regulation is that the specific site of transgene insertion into the host genome cannot be targeted and will therefore be unique for each transformant. Because such a random inser-tion could interfere with the normal functioning of endogenous genes, with potentially harmful unintended effects, regulators presume that each event may be uniquely risky. However, there is no evidence that the uncertainties associated with transgene insertion are any greater than those that occur with other forms of genetic modification, such as the random genetic changes that result from mutation breeding, the pleitropic effects on gene structure and expression common in wide crosses and ploidy modification, or even those

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that accompany the movement of transposable elements in normal sexual reproduction (Weber et al., 2012; Steiner et al., 2013). Yet, while all breeding methods pose a theoretical risk of unintended genetic changes that could result in an increase in toxins and other harmful substances, or a reduction in dietary nutrients or other beneficial constituents, such effects are routinely identified by basic phenotype analysis. In fact, as Bradford et al. observe, “con-ventional breeding programs generally evaluate populations with much wider ranges of phenotypic variation than is observed in transgenic programs” (2005, p. 441), and they successfully eliminate potentially harmful plants from devel-opment programs. With the advent of inexpensive methods of DNA sequenc-ing, the site of such gene insertion events is now always well characterized.

It is also commonly believed that transgenic crops should be regulated because they express novel traits that not only are not normally associated with that crop, but which in many cases have not been part of the human or animal diet. When a genuinely novel substance (e.g., a new protein or other phytochemical) is introduced into a plant, this does merit special testing to assure the crop is safe for consumers and the environment. But most of the traits introduced into biotech crops currently on the market can also be introduced with various classical breeding methods.

For example, herbicide tolerance, the most widely adopted transgenic trait, is routinely introduced into crop species via selection or mutation breeding. And while the transgene responsible for tolerance to the herbicide glyphosate, a common trait in biotech plants, was isolated from the common soil microbe Agrobacterium tumefaciens, the wild type gene and the EPSP synthase protein for which it codes can be detected in many non-transgenic food crops due to the presence of soil residues in harvested crops. Furthermore, all plants in the human diet naturally contain a gene that encodes an EPSP synthase protein that is required for normal plant growth and development. All transgenic insect resistant plants commercialized to date contain a gene from one of several subspecies of naturally occurring soil bacterium Bacillus thurengiensis (Bt). But whole Bt spores have long been cultured for use as a “natural” insecticide for food and ornamental crops, so consumers have a long history of exposure to Bt genes and the specific proteins responsible for transgenic insect resistance. In fact, thuricides such as DIPEL made from Bt spores are the only insecticides approved for use by the organic food industry. Moreover, various classical breeding methods, such as interspecific and intergeneric “wide cross” hybridization, frequently introduce new genes and gene products into the human diet. Thus, not all biotech plant varieties contain genes or proteins new to the food supply, nor is the introduction of novel substances unique to transgenic breeding methods.


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It is worth repeating that the only scientific justification for pre-market safety assessment for any new plant variety is to establish the safety of any newly introduced substances. It is an unfortunate reality that pre-market safety assessment has become an endless search for unintended effects which have become like the new clothes in fable of the “Emperor’s New Clothes.” The risk assessment described in the following paragraph has been applied to accomplish the assessment.

Risk assessmentThe consensus of scientific opinion and evidence is that the application

of GE technology introduces no unique food/feed safety or environmental impact concerns and that there is no evidence of harm from those products that have been through a regulatory approval process. This conclusion has been reached by numerous national and international organizations (e.g., Food and Agriculture Organization/World Health Organization of the United Nations, Organization for Economic Cooperation and Development, EU Commission, French Academy of Sciences, National Research Council of the National Academy of Sciences, Royal Society of London and Society of Toxicology).

In contrast to traditionally bred crops, a rigorous safety-testing paradigm has been developed and implemented for GE crops, which utilizes a system-atic, stepwise and holistic safety assessment approach (Cockburn, 2002; Kok and Kuiper, 2003; König et al., 2004). The resultant science-based process focuses on a classical evaluation of the toxic potential of the introduced novel gene, its gene product, and the wholesomeness for human consumption of the GE crop. In addition, detailed consideration is given to the history and safe use of the parent crop as well as that of the gene donor(s). The overall safety evaluation is conducted using the process known as “substantial equivalence (SE),” a model that is entrenched in all international crop biotechnology guidelines (Kok and Kuiper, 2003; Codex, 2003). The SE paradigm provides the framework for a comparative approach to identify the similarities and differences between the GE product and an appropriate comparator that has a known history of safe use. By building a detailed profile on each step in the transformation process (from parent to new crop) and by thoroughly evaluat-ing the significance, from a safety perspective, of any differences that may be detected between the GE crop and its comparator, a comprehensive matrix of information is constructed. This information is used to reach a conclusion about whether food or feed derived from the GE crop is as safe as food or feed derived from its traditional counterpart or the appropriate comparator.

One common misunderstanding of the GE plant testing process involves the meaning and role of substantial equivalence. Some biotechnology critics have claimed that regulatory authorities have deemed all GE plants to be ipso facto substantially equivalent to their conventional counterparts, thereby

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requiring no safety testing at all. But as we describe above, the substantial equivalence concept merely describes the way in which GE plants should be tested. A conclusion that a product is substantially equivalent can only be reached after it is rigorously compared to a non-GE counterpart for material differences. Yet, even when a material difference is detected and the GE product is found to be not substantially equivalent to its conventional com-parator, the finding does not automatically mean that the product is unsafe. Additional testing that explores these differences more completely may ultimately determine that the differences have no bearing on the product’s safety for consumers or the environment.

Using this approach in the evaluation of more than 90 GE crops that have been approved in the U.S., the conclusion has been reached that foods and feeds derived from GE crops are as safe and nutritious as those derived from tradi-tional crops. The lack of any credible reports of adverse effects resulting from the production and consumption of GE crops grown on more than 235 million cumulative hectares over the last seven years supports these safety conclusions.

The U.S. National Research Council in “Genetically Modified Pest-Protected Plants: Science and Regulation” (NRC, 2000) determined that no difference exists between crops modified through modern molecular techniques and those modified by conventional breeding practices. The NRC emphasized that the authors were not aware of any evidence suggesting foods on the market today are unsafe to eat because of genetic modification. In fact, the scientific panel concluded that growing such crops could have environ-mental advantages over other crops.

In a 2003 position paper, the Society of Toxicology (SOT, 2003) cor-roborated this finding and noted that there is no reason to suppose that the process of food production through biotechnology leads to risks of a different nature than those already familiar to toxicologists or to risks generated by conventional breeding practices for plant, animal or microbial improvement. It is therefore important to recognize that it is the food product itself, rather than the process through which it is made, that should be the focus of atten-tion in assessing safety.

Similarly an EU Commission Report (EU, 2001, 2008) that summarized biosafety research of 400 scientific teams from all parts of Europe conducted over 15 years stated that research on GE plants and derived products so far developed and marketed, following usual risk assessment procedures, has not shown any new risks to human health or the environment beyond the usual uncertainties of conventional plant breeding. Indeed, the use of more precise technology and the greater regulatory scrutiny probably make GE plants even safer than conventional plants and foods. More recently the Commission


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funded research from 130 research projects involving 500 independent research groups over 25 years, concluding that “There is, as of today, no scientific evidence associating GE with higher risks for the environment or for food and feed safety than conventional plants and organisms” (Europa Press Release, 2010). Recent transcriptomic and metabolomic (Baker, 2006; Catchpole, 2005; Ricroch et al., 2011) studies in wheat and potatoes respec-tively show greater variation within and between conventionally bred culti-vars and even growth locations than between GE and parental variety, except for the intended change. In fact, the differences within the same line between different geographical sites were generally greater than differences between various control and test lines at the same site.

The development of new tools for “omic” analysis (e.g., transcriptomics, proteomics and metabolomics) has prompted their evaluation for the safety assessment of transgenic crops since these kinds of untargeted holistic analytical methods offer the possibility of a more comprehensive insight into gene expression, protein content and detailed composition (Chassy, 2010). Although “omic” technologies have proven to be a powerful research tools, the application of these methods offers no useful information to the safety assessor. Transcriptomics is of limited value since measuring changes in gene expression cannot be translated directly into an understanding of changes in risk, or in metabolic or phenotypic traits. There are associated methodological, data handling and bias problems associated with transcriptomics as well and there is a paucity of baseline data that can be used to establish normal ranges. At this point in time, and for the foreseeable future, it is better to directly measure the outcome of genotype x environment interactions instead of trying to predict potential side effects based on observed changes in gene expression.

Proteomic analysis suffers from a similar set of problems (Chassy, 2010). Only a few hundred of the thousands of potential proteins in a cell can be evaluated; few can be evaluated with quantitative precision. More impor-tantly, as is the case with transcriptomic analysis, estimation at a semi-quan-titative level of which proteins are present in a cell, organ, tissue or organism, is not predictive of the composition of the organism, the phenotype of the organism, or any changes in risk associated with consumption of the organism. Proteomic analysis may, however, be useful as a tool for the rapid targeted analysis of specific proteins or sets of proteins as, for example, in the evaluation of changes in the content of allergens in a plant known to cause allergy.

The application of metabolomic profiling is more attractive from a safety assessment perspective since it focuses on the composition of the plant food or feed that is actually consumed (Chassy, 2010). Metabolomic profiling can be used for sample discrimination and classification (e.g., for GE comparator analysis), or for biochemical and mechanistic studies in discovery research

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and development. These tools may someday be employed to understand which parts of larger biochemical networks respond to genetic modification. While it has been claimed that unbiased non-targeted and comprehensive as-sessments can be improved by metabolite analyses, the usefulness of metabo-lomics as a safety assessment tool suffers from the same set of limitations that apply to transcriptomics and proteomics. At present, metabolomics is not one technology, but a family of analytical techniques each of which is capable of detecting the presence of tens to hundreds of metabolites. The data are difficult to reproduce; they are not quantitative; and baseline data and normal ranges for metabolites are not available. Moreover, it is often the case that many of the compounds detected are of unknown structure.

It is worth noting with regard to the usefulness, or lack of usefulness, of omic profiling for safety assessment, that the currently employed targeted analytical paradigm focuses on the measurement of all known macro- and micronutrients, toxicants, anti-nutrients, and phytochemicals of special interest in each specific crop (Harrigan and Chassy, 2012). The analysis often accounts for 95 percent or more of the composition of a sample. There is no evidence that targeted analysis is inadequate in identifying changes in composition that could be of concern to the risk assessor. Furthermore, it has been found that cultural, environmental and geographic differences often lead to large differences in composition between test samples of the same variety and these differences are often greater than those observed between different varieties of the same crop. Recall that the composition of transgenic crops more closely resembles that of their parental varieties than does that of other varieties of the same crop to the parental strain and to one another (Ricroch et al., 2011). At this point in time, omic profiling is not necessary to establish comparative safety, nor are standardized and validated omic methods ready for application in safety assessment.

The main principles of the international consensus approach to safety assess-ment of transgenic crops are listed below (Chassy et al., 2004; Chassy et al., 2008; Ricroch et al., 2011). They serve to illustrate the variety of principles that have been at the center of the discussions and that are continuously being updated:

□ Substantial equivalence: This is the guiding principle for safety assess-ment. In short, substantial equivalence involves the process of comparing the GE product to a conventional counterpart with a history of safe use. Such a comparison commonly includes agronomic performance, phenotype, expression of transgenes and composition (macro- and micronutrients), and it identifies the similarities and differences between the GE product and the conventional counterpart. Based on the differences identified, further inves-tigations may be carried out to assess the safety of these differences. These assessments include any protein(s) that are produced from the inserted DNA.


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□ Potential gene transfer: Where there is a possibility that selective advantage may be given to an undesirable trait from a food safety perspec-tive, this should be assessed. An example is in the highly unlikely event of a gene coding for a plant made pharmaceutical being transferred to corn. Where there is a possibility that the introduced gene(s) may be transferred to other crops, the potential environmental impact of the introduced gene and any conferred trait must be assessed.

□ Potential allergenicity: Since most food allergens are proteins, the potential allergenicity of newly expressed proteins in food must be con-sidered. A decision-tree approach introduced by ILSI/IFBC in 1996 has become internationally acknowledged and recently updated by Codex FAO/WHO, 2003. The starting point for this approach is the known allergenic properties of the source organism for the genes. Other recurrent items in this approach are structural similarities between the introduced protein and allergenic proteins, digestibility of the newly introduced protein(s), and eventually (if needed), sera-binding tests with either the introduced protein or the biotechnology-derived product.

□ Potential toxicity: Some proteins are known to be toxic, such as en-terotoxins from pathogenic bacteria and lectins from plants. Commonly employed tests for toxicity include bioinformatic comparisons of amino acid sequences of any newly expressed protein(s) with the amino acid sequences of known toxins with those of introduced proteins, as well as rodent toxicity tests with acute administration of the proteins. In addition to purified proteins, whole grain from GE crops has been tested in animals, commonly in subchronic (90-day) rodent studies.

□ Unintended effects: Besides the intended effects of the modification, interactions of the inserted DNA sequence with the plant genome are possible sources of unintended effects. Another source might be the introduced trait unexpectedly altering plant metabolism. Unintended effects can be both predicted and unpredicted. For example, variations in intermediates and endpoints in metabolic pathways that are the subject of modification while undesirable are predictable, while switching on/expression of unknown endogenous genes through random insertion in regions of the genome that control gene expression is both unintended and unpredictable. The process of product development that selects a single commercial product from hundreds to thousands of initial transformation events eliminates the vast majority of situations that might have resulted in unintended changes. The selected commercial product candidate event undergoes additional detailed phenotypic, agronomic, morphological and compositional analyses to further screen for such effects.

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□ Long-term effects: It is acknowledged that the premarket safety assess-ment should be rigorous to exclude potentially adverse effects of consump-tion of foods or feeds derived from GM crops. Nevertheless, some have insisted that such foods should also be monitored for long-term effects by postmarket surveillance. No international consensus exists as to whether such surveillance studies are technically possible without a testable hypoth-esis in order to provide meaningful information regarding safety, and a GM crop with a testable safety concern would most likely not pass regulatory review. The notion of using measurable biomarkers has been suggested but then these need to be determined for all foods and feeds whatever the source and balanced against reasonable economic burden.

The question of whether foods derived from organisms modified with recombinant DNA techniques should be specially labeled has received a great deal of attention. The FDA’s (1992) approach to the labeling of foods, includ-ing those genetically engineered or otherwise novel, is that the label must be accurate and “material.” Agency officials recognized that any breeding method could impart a change that makes food less safe or nutritious than its conventional counterpart, but that the process of rDNA modification is not inherently risky. Accordingly, special labeling is required “if a food derived from a new plant variety differs from its traditional counterpart such that the common or usual name no longer applies, or if a safety or usage issue exists to which consumers must be alerted.”

Such changes include the introduction of a toxin, antinutrient or allergen into a food product in which consumers would not ordinarily expect to find it, such as an allergenic protein from nuts in corn; the elevation of an endog-enous substance to potentially harmful levels, such as a significant increase in potato or tomato glycoalkaloids; or a significant change in the level of dietary nutrients in a food, such as oranges with abnormally low levels of vitamin C or a significant change in the lipid composition of cooking oils. Other material changes that must be labeled include those that relate to the storage, prepara-tion or usage characteristics of a food, such as a change affecting the length of time or manner in which kidney beans must be soaked and cooked before eating or the safe shelf-life of various food products. Even a change in organoleptic characteristics of a food from what consumers would normally expect, includ-ing the taste, smell or mouth feel of a food, is considered material and must be labeled. Importantly, the FDA’s policy stipulates that the altered characteristic itself must be specified on the label, not the breeding method used to impart the change. In that regard, labeling serves to alert consumers to the new character-istic of foods, not the processes used in their development.

The FDA’s policy statement also emphasizes that no premarket review or approval is required unless characteristics of the biotech food explicitly


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raise safety issues, and that – in as much as the genetic method used in the development of a new plant variety does not meet either of the two criteria for “materiality” – the FDA cannot require the labeling to include this informa-tion. Obviously, many of the novel nutritionally enhanced foods expected on the market in the next few years will be labeled, as they will differ from their traditional counterparts, and in most instances, the company marketing them will want to proclaim their enhanced nutritional value.

Applications overview4Modifications of crop plants can be organized into two broad, non-mutual-

ly exclusive categories: those that benefit the producer through introduction of properties such as improved insect, weed and disease management and lower input costs, and those that benefit the consumer more directly with increased nutritional value, flavor or other desirable functional product attributes. Many plants in both categories also, either directly or indirectly, deliver benefits for the environment, such as reducing insecticide use and hastening an ongoing shift to conservation tillage practices. Modifications that protect the crop from either biotic or abiotic stress or that increase total crop yield primarily benefit the producer and are often called “input traits.” (Biotic stress is damage by predators, such as insects and nematodes; weeds; or disease agents, such as viruses, fungi, and bacteria. Abiotic stress is damage from other, usually physical or climatic, causes, such as drought, flooding, cold, heat, salination and poor soils.) Scientists have just begun to tap the poten-tial of biotechnology to produce varieties of plants that confer advantages to consumers directly. Varieties modified to have greater appeal to consumers are said to have enhanced “output traits.” The majority of crops produced using modern biotechnology in present day commercial use fit in the former group.

In the United States, which has the largest number of approved and commercially planted biotech varieties, the U.S. Department of Agriculture has approved (in agency parlance, “deregulated”) more than 90 transformation events of 16 plant species for commercial-scale cultivation (see http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml) – though many of these products, while legal to grow and sell, are not commercially available. U.S. farmers also grow the largest number of acres (over 150 million) with biotech varieties, followed by Brazil (67m), Argentina (53m), India (23m), and Canada (23m). But by 2012, 28 countries had approved at least one transformation event for commercial cultivation, and these crops were grown by more than 17 million farmers on over 420 million acres total (James, 2013). Twenty of the 28 coun-tries are emerging economies, and a full 90 percent of the 17.3 million farmers are resource-poor farmers from LDCs. Another 31 countries have permitted

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Applications Overview 4

pre-commercial field trials with biotech crop varieties, or have approved some harvested biotech plants to be imported for use as food and livestock feed.

Among the varieties that are currently marketed, the most common traits are insect resistance, herbicide tolerance and virus resistance. And the most widely adopted species are the commodities corn, cotton, soy and canola. U.S. farmers grew each of these crops and planted a significant number of acres with biotech varieties of sugar beet and alfalfa, while a far smaller number of acres were planted with biotech squash, papaya and rice.

Among the most prevalent first generation products of agricultural biotechnol-ogy have been crop varieties resistant to certain chewing insects. This pest-resis-tance trait was added by inserting a gene from the common soil bacterium Bacillus thuringiensis (Bt), which produces an insoluble crystalline protein that adheres to and degrades the alkaline stomach of insect larvae, causing death. Different strains of the Bt bacterium produce proteins that are toxic to a specific range of insects, but not to mammals, fish, birds or other animals, including humans (EPA 2001). The bacterial proteins occur naturally, and organ

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